少突胶质细胞系基因-髓鞘碱性蛋白在口腔扁平苔藓中表达的研究

目的 通过观察少突胶质细胞系基因-髓鞘碱性蛋白(Golli-MBP)在口腔扁平苔藓(OLP)患者外周血单个核细胞中的表达,探索OLP的发病机制.方法 采用密度梯度离心法分离20例充血糜烂型、16例光滑型OLP患者,以及19例对照组的外周血单个核细胞,分别用逆转录-聚合酶链反应(RT-PCR)法和Western blot...     

少突胶质细胞系基因-髓鞘碱性蛋白在口腔扁平苔藓中表达的研究

目的 通过观察少突胶质细胞系基因-髓鞘碱性蛋白(Golli-MBP)在口腔扁平苔藓(OLP)患者外周血单个核细胞中的表达,探索OLP的发病机制.方法 采用密度梯度离心法分离20例充血糜烂型、16例光滑型OLP患者,以及19例对照组的外周血单个核细胞,分别用逆转录-聚合酶链反应(RT-PCR)法和Western blot法检测3组研究对象外周血单个核细胞中Golli-MBP mRNA和蛋白的表达.结果 充血糜烂型和光滑型OLP患者中Golli-MBP mRNA和蛋白的表达水平均高于对照组,且差异有统计学意义(P＜0.01);但充血糜烂型和光滑型OLP患者组间表达的差异均无统计学意义(P＞0.05).结论 Golli-MBP在OLP的发病过程中起着重要作用.     

少突胶质细胞谱系基因-髓鞘碱性蛋白在口腔扁平苔藓组织中的表达

目的：探讨少突胶质细胞谱系基因一髓鞘碱性蛋白（genes of the oligodendrocyte lineage—myelin basic protein,Golli—MBP）在口腔扁平苔藓（orallichenplanus,OLP）组织中的表达及其与OLP发病的关系。方法：采用实时荧光定量PCR的方法,检测39例OLP患者病损组织和16例健康口腔黏膜组织中Golli—MBP的基因表达水平,采用免疫组化法检测38例OLP及20例正常口腔黏膜组织石蜡标本中Golli—MBP的表达情况。结果：Golli一删即mRNA在OLP组中的表达显著高于正常对照组（P〈0．001）。OLP患者石蜡标本中Golli—MBP的表达水平亦显著高于对照组（P〈0．001）;固有层淋巴细胞中Golli—MBP的表达高于对照组（P〈0．05）;上皮细胞中Golli—MBP的表达在OLP患者及对照组间无统计学差异（P〉0．05）。结论：Golli—MBP在OLP组织中高表达,可能在OLP的发病机制中起一定作用。     

慢病毒介导过表达端粒酶逆转录酶的 人口腔黏膜上皮稳定细胞系的构建

目的 通过慢病毒法建立稳定表达外源性人端粒酶逆转录酶（hTERT）基因的口腔黏膜上皮细胞（OMECs）,探索构建高效、稳定的永生化OMECs细胞系的方法。方法 提取293T细胞总RNA,应用聚合酶链反应（PCR）法扩 增hTERT基因全长,构建重组慢病毒载体pLVX-puro-hTERT。包装慢病毒颗粒后感染人正常OMECs,经嘌呤霉素抗 性筛选获得阳性克隆,采用实时荧光定量PCR法和Western blot法检测hTERT基因mRNA和蛋白的表达水平。结果 成功构建了pLVX-puro-hTERT过表达慢病毒载体并感染到OMECs中;感染细胞与正常OMECs形态相似,呈铺路石 样生长;实时荧光定量PCR和Western blot结果均显示,hTERT在感染细胞中高表达,与正常细胞相比差异有统计学 意义（P<0.05）。结论 通过慢病毒法成功建立了过表达hTERT的OMECs稳定细胞系,为构建高效、稳定增殖的人 永生化OMECs细胞系奠定了实验基础。     

慢病毒载体感染人口腔黏膜成纤维细胞稳定表达绿色荧光蛋白的研究

目的：确立慢病毒载体高效感染人口腔黏膜成纤维细胞的感染条件,为进一步应用基因工程技术研究人口腔黏膜成纤维细胞奠定基础。方法：采用组织块法培养原代人口腔黏膜成纤维细胞,免疫组化SP法鉴定细胞类型,将携带绿色荧光蛋白基因的慢病毒载体以不同感染复数（multiplicity of infection,MOI）感染纯化细胞,并设置不同感染条件,感染4 d后于荧光显微镜下观察绿色荧光蛋白表达情况。结果：成功分离纯化得到人口腔黏膜成纤维细胞,当慢病毒感染复数为30,polybrene浓度为8μg/mL,并加入感染增强液时,可达到较高的感染效率,满足后续实验需要。结论：慢病毒载体可高效感染人口腔黏膜成纤维细胞,携带的绿色荧光蛋白基因可在成纤维细胞内稳定表达。     

生长分化因子15基因过表达慢病毒载体的构建

目的 构建生长分化因子15（growth differentiation factor 15,GDF15）基因过表达慢病毒载体。 方法 根据GDF15 mRNA的基因序列,合成GDF15基因,构建至过表达载体并转化至感受态细胞,再进行测序验证。重组慢病毒载体经过测序鉴定后,转染293T细胞,包装生产慢病毒。用慢病毒转染293T细胞,再用Western blot检测GDF15的表达情况。 结果 测序结果证实GDF15 基因正确插入载体中。慢病毒转染293T细胞后,GDF15基因在蛋白质水平上表达显著增加。 结论 GDF15基因过表达慢病毒载体构建成功,并有效增强GDF15基因在293T细胞中的表达。     

microRNA-223过表达与抑制表达 慢病毒载体的构建及鉴定

目的 构建 microRNA-223过表达慢病毒载体及抑制表达慢病毒载体,并验证其过表达或抑制表达microRNA-223的效率。方法 设计针对microRNA-223前体的过表达基因片段及针对microRNA-223成熟体的反义片段,应用聚合酶链反应（PCR）调取目的基因及通过退火反应获得双链DNA寡聚核苷酸片段。并通过基因重组技术将目的 基因片段及反义片段分别克隆到GV229及GV232慢病毒载体中,进行PCR鉴定及DNA测序比对分析,构建相应的 microRNA-223过表达慢病毒载体及抑制表达慢病毒载体。利用荧光定量PCR法检测microRNA-223表达水平。结果 对阳性克隆进行PCR鉴定及DNA测序证明,microRNA-223过表达慢病毒载体及抑制表达慢病毒载体构建成功。microRNA-223过表达慢病毒载体能显著提高细胞中microRNA-223表达水平,microRNA-223抑制表达慢病毒载体能明显抑制细胞中microRNA-223表达水平。结论 成功构建microRNA-223过表达慢病毒载体及抑制表达慢病毒载体,为进一步研究microRNA-223对口腔癌发生发展的影响及其机制提供了稳定的细胞转染载体。     

慢病毒表达载体pLentiTrident1-hBMP2-Neo-hNGF的构建及鉴定

目的:构建并鉴定含有人骨形成蛋白2(hBMP2)及神经生长因子(hNGF)目的基因的慢病毒载体.方法:使用限制性内切酶,将新霉素抗性基因(Neo)从载体pLentiTrident1-EGFP-Neo切下,插入pLentiTrident1空载体的相应多克隆位点上.构建出含新霉素抗性的表达载体.采用PCR扩增得到hBMP2与hNGF的目的片段,通过多克隆位点将2个目的基因插入表达载体.对该重组载体进行酶切鉴定、PCR鉴定以及DNA序列测序分析.结果:通过酶切鉴定及测序分析,慢病毒表达载体(plentiTrident1-hBMP2-Neo-hNGF)构建成功.结论:成功构建了hBMP2与hNGF的共表达慢病毒载体.为研究神经因素在骨组织再生中的作用奠定了基础.     

人Notch-1基因RNA干扰慢病毒载体的构建及鉴定

目的 构建及鉴定人Notch-1基因的RNA干扰（RNAi）慢病毒载体,寻找最佳RNAi慢病毒载体。方法 针对人Notch-1基因序列,按照RNAi序列设计原则,设计3段RNAi靶点序列（shRNA1~3）,通过限制性内切酶BamHⅠ和EcoRⅠ双酶切、T4 DNA连接酶连接,将Notch-1基因序列插入慢病毒载体pLenOR-THM,构建pLenOR-THM-Notch-1重组载体。质粒转化感受态DH5α细菌,筛选阳性克隆,经KpnⅠ和EcoRⅠ双酶切及测序鉴定正确后通过脂质体将慢病毒四质粒系统共转染293T细胞,进行慢病毒包装并测定病毒滴度,观察感染效率。各组病毒载体转染ACC-M细胞后,运用定量逆转录聚合酶链反应和Western blot检测Notch-1基因mRNA和蛋白的表达水平。结果 成功构建慢病毒载体pLenOR-THM-Notch-1,四质粒共转染293T细胞后可见大量绿色荧光;浓缩后的病毒滴度为5.8×108 TU·mL-1;以复感染系数为1时感染293T细胞,感染效率在90％以上。QRT-PCR和Western blot检测结果表明,pLenOR-Notch-1-shRNA3组受抑制程度最高。结论 成功构建了人Notch-1 RNAi慢病毒载体。     

E2F-1基因RNAi慢病毒载体的构建与鉴定

目的:构建和鉴定靶向E2F-1基因的RNA干扰慢病毒载体.方法:体外合成两对互补并编码靶向E2F-1基因的特异性短发夹RNA(short hairpin RNA,shRNA)序列和一个阴性对照组的寡核苷酸,克隆到pLKO.1-PURO-U6(Age I/EcoR I)质粒载体中,经酶切和测序鉴定所构建的重组载体是否正确...     

口腔扁平苔藓患者血硒水平的测定及初步研究

目的探索口腔扁平苔藓与必需微量元素硒的关系。方法30例口腔扁平苔藓实验组与30例健康对照组,采用原子吸收分光光度法测定红细胞及血清硒水平。结果实验组红细胞及血清硒水平均低于健康对照组（P＜0.05）。结论硒水平低下可能是口腔扁平苔藓发病原因之一。     

Oral lichen planus: a preliminary clinical study on treatment with tazarotene

OBJECTIVE: The rationale for using tazarotene in oral lichen planus (OLP) is its regulatory action on the growth and differentiation of keratinocytes and on inflammation. This randomized, placebo-controlled study addresses evaluation of the effects of topic tazarotene in the treatment of OLP. DESIGN: The degree of lesions before and after treatment scored by a 6-score scale in six cases treated with tazarotene was statistically compared with those of six controls treated with placebo. SUBJECTS: Twelve patients with hyperkeratosic OLP were randomly allocated to treatment with tazarotene gel 0.1% b.i.d. or with placebo for eight consecutive weeks. METHODS: The statistical comparison was executed by means of Wilcoxon analysis for paired data. RESULTS: Patients treated with tazarotene presented a significant reduction of their lesions as compared with the control group. Among transitory side-effects, burning sensation and taste abnormalities were observed. CONCLUSION: Topical tazarotene may be a valuable therapeutic tool in the treatment of hyperkeratotic OLP.     

A clinical study of 674 patients with oral lichen planus in China

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease with different clinical presentations that can be classified as reticular, atrophic or erosive. Although OLP is a relatively common disorder, the reports comprising large numbers of OLP patients with specific character are lacking in the literature. The purpose of this paper was to describe the clinical characteristics of OLP in 674 Chinese patients. METHODS: A total of 674 charts of patients with histologically confirmed OLP were collected from Stomatological Hospital of Wuhan University between 1963 and 2003. RESULTS: Of the 674 patients, 65.9% were women and 34.1% were men. The most common clinical presentation was the reticular type (51.3%), and symptomatic OLP was noted in 67.5% of the patients, mainly in those with the erosive form. The erosive presentations showed significantly longer duration, more sites affected and a much greater old patients predominance than reticular or atrophic ones. About 90.9% of the patients had multiple oral sites of involvement and isolated lower lip lichen planus were observed in 60 cases (8.9%) and isolated gingiva lichen in only one case (0.2%). Skin involvement of lichen planus was found in 11.4% of patients. No statistically significant differences could be identified between OLP and diabetes, cardiovascular disease, smoking or alcohol use. Precipitating factors that resulted in an exacerbation of the disease were frequently noted and included foods, stress, dental cusp and poor oral hygiene. The transformation of OLP into malignancy was observed in four patients at sites previously diagnosed by clinical examination as erosive or atrophic lichen planus. CONCLUSIONS: Patients with OLP in China usually present with distinctive clinical morphology and characteristic distribution and few may display lesions with a confusing array of forms mimicking other diseases. A long time follow up is of utmost importance to detect its malignant transformation.     

Oral lichen planus: a retrospective study of 690 British patients

OBJECTIVE: This is the largest UK patient group with oral lichen planus (OLP) to be studied in terms of the demographic and clinical characteristics. MATERIALS AND METHODS: Data were taken from the medical records of 690 consecutive patients referred to Oral Medicine subsequently found to have clinical, and usually histopathological confirmatory features of OLP. Over two-thirds (68.7%) of the patients were Caucasians. RESULTS: Eighty-two per cent of the patients had been referred to a specialist Oral Medicine service by general dental practitioners, 62% of the patients being referred as a consequence of oral mucosal and/or gingival pain. Reticular OLP was the most common intra-oral presentation, but 60% of such lesions were accompanied by other clinical types of OLP. 95% of lesions were bilateral. About 13% of patients reported symptoms or signs, or had a known history of lichen planus or possible lichen planus affecting non-oral epithelia. In only 13% of patients did all signs and symptoms of OLP resolve within 12-246 months (median 35 months). A malignant transformation rate of 1.9% was observed in the present group. CONCLUSIONS: Oral lichen planus in UK persons almost always gives rise to bilateral reticular OLP, rarely resolves spontaneously, and has a low rate of malignant transformation.     

白介素12A基因多态性与OLP的相关性研究

目的：研究华东地区汉族人群IL-12A基因多态性与口腔扁平苔藓（orallichenplanus,OLP）的相关性。方法：采用TaqMan荧光定量PCR法检测华东地区292例OLP患者及686例正常对照者的IL-12A基因的5个SNP位点（rs3024415,rs2243123,rs583911,rs568408和rs2243143）,分析IL-12A基因多态性与OLP的相关性。结果：①华东地区健康人群IL-12A基因3′-UTRrs568408位点AA、GA和GG基因型频率分别是0．7％、12．4％和86．8％,而OLP患者分别是1．1％、18．4％和80．5％,其中GA和AA基因型分布频率显著高于正常对照组@=O．0427）。②正常对照组IL-12A基因rs583911位点AA、GA和GG基因型频率分别是4．2％、38．6％和57．1％,而OLP患者分别是9．5％、36．5％和54％,其中AA基因型分布频率显著高于对照组（p=O．00805）。③糜烂型OLP组IL-12A—rs568408／A等位基因频率显著高于正常对照组,分别为11．6％和6．94％（OR=I．76,95％CI：1．133—2,732,P=0．011）。结论：华东地区汉族OLP人群IL-12A基因rs568408位点存在多态性变异,可能与OLP的疾病易感性和严重程度有关。     

Periodontal status in patients with oral lichen planus: a study of 90 cases

OBJECTIVE: This study was carried out to relate periodontal status to the oral lesions in patients with oral lichen planus (OLP). MATERIALS AND METHODS: Periodontal status was evaluated in 90 patients with OLP and in 52 controls, in terms of the plaque index (PII), simplified calculus index (CIS) and periodontal disease index (PDI). RESULTS: No significant differences were observed between the two groups as regards the different periodontal indices. The plaque and calculus indices were higher in the more extensive forms of OLP (P = 0.02 and P = 0.012, respectively), and in the presence of gingival involvement (P = 0.004 and P = 0.04). A significant association was also observed between the presence of atrophic-erosive lesions and increased periodontal deterioration (P = 0.037). CONCLUSIONS: Increased plaque and calculus deposits are associated to a significantly higher incidence of atrophic-erosive gingival lesions in patients with OLP.